[unreadable] We propose a strategy for systematic functional annotation of cis-regulatory sequences in a metazoan genome. We will combine automated comparative DNA sequence analysis and comparative gene expression analysis to identify regulatory elements in Drosophila. Our laboratory is engaged in a project to provide functional annotation of the Drosophila melanogaster genome using gene expression profiling during development. Here we propose to utilize the D. pseudoobscura genome for comparative gene expression profiling and regulatory motif analysis. We will test whether combining comparative sequence analysis with comparative gene expression analysis will allow us to systematically decode regulatory information in the genome. Regulatory elements identified in our studies will be tested experimentally in vivo. Our results will also identify sets of genes that have evolved expression. Regulatory elements associated with gene expression evolution will be examined in order to better understand the functional consequences of cis-regulatory sequence evolution. Principles derived from the proposed studies will likely be useful for interpreting the functional significance for conserved and diverged regulatory sequences in other genome comparisons, including those of primates and other vertebrates. [unreadable] [unreadable]